人肝门部胆管癌裸鼠肝门移植模型的建立及CD、HSV-tk双自杀基因治疗实验研究

【作者】 李志伟；

【导师】 王占民；

【作者基本信息】 山东大学 ， 普外科， 2004， 博士

【摘要】 目的 建立人肝门部胆管癌裸鼠肝门移植模型,对其生物学特性进行初步探讨。 方法 收集第90代肝门部胆管癌细胞系FRH细胞(2×10~6)接种于6只Balb/c裸鼠后肢背侧皮下,两周左右,有5只Balb/c裸鼠后肢背侧皮下移植瘤长至约8mm×6mm,切取裸鼠皮下移植瘤边缘直径约1mm组织块植入Balb/c裸鼠肝门部肝实质内,共32只,观察裸鼠生活情况。每周取两只裸鼠在麻醉下剖腹探查,观察肝门部肿瘤生长状况,测量肿瘤直径,计算肿瘤体积,绘制裸鼠肿瘤生长曲线。待裸鼠出现黄疸,全身衰竭时,予以处死,抽血检查肝功能;解剖肝门部肿瘤及观察周围浸润情况,有、无远处脏器转移并行病理检查。裸鼠肝门部移植瘤与人肝门部胆管癌标本进行比较,观察肿瘤病理结构;透射电镜观察肿瘤细胞超微结构;免疫组化检测VEGF、bFGF、PCNA、bcl-2、Fas、P53、P21、MMP-9、TIMP-1、TIMP-2、CD34表达情况,测定肿瘤微血管密度(MVD),。 结果 32只裸鼠建成30只肝门部肿瘤移植模型,成瘤率94%;建成的30只模型中28只出现黄疸,黄疸出现率为93%。接种后2周左右可触及肝门部有小结节,平均4周左右出现黄疸,6周左右裸鼠开始出现恶液质。解剖发现肝门部有肿瘤生长,肿瘤生长较快,长径1.8～2.8cm,包膜不完整,向周围组织浸润。主要向肝实质内生长,8例侵及胆囊,6例浸及肝门血管,3例腹腔淋巴结转移。肾脏、脾脏、胃肠、肺脏未见转移灶。肝功能检查显示:ALT 86±28.7U/L,AST 98±31.3U/L,GGT 122±41.6U/L,ALP 168±65.8U/L,TBIL 35±13.6μmol/L,DBIL 31±11.5μmol/L,均明显高于正常对照组。肝门肿瘤HE染色呈低分化胆管腺癌表现,与人肝门部胆更多还原

【Abstract】 OBJECT To establish a xenograft model in porta hepatis of human hilar cholgangiocarcinoma in nude mice, and observe its biological characteeristics. METHOD The 90^th generation FRH cells（2 × 10⁶） were subcutaneously transplanted into the back of the hindlimbs of 6 Balb/c-nu/nu mice. When the tumors reached 8mm × 6mm in diameter two weeks late, the tumors were cut into 1mm³ pieces and retransplanted into porta hepatis of 32 new nude mice.The mice were observed every day. Each week the belly of two mice were laparotomize to observe the growth of xenograft tumor by measuring its diameter and volum, and the growth curves of the tumor were figured. The mice were killed when jaundice appeared and they were on the brink of death.They were laparotomized to exmine metastatic tumors in nearby and distants organs and pathologic examanition was made. Electron microscope was used to observe the ultrastructure. VEGF, bFGF, PCNA, bcl-2, Fas , P53, P21, MMP-9, TIMP-1, TIMP-2, CD34were examined with imrnunohistochemical methods. Microvessel density （MVD） was counted also to observe the expression of these tumor marks.RESULT 30 out of 32 nude mice xenograft model of human hilar cholangiocarcinoma were successfully established. The rate of tumor occurring was 94%.28 out of 30 nude mice had , The rate of jaundice occurring was 93%. About two weeks later, small nodes in porta hepatis were found; and about four weeks later, jaundice occurred. Dyscrasia appeared in six weeks or so. The xenograft tumor reached 1.8 to 2.8cm in major diameter.更多还原